Listeria monocytogenes post-outbreak management - When could a food production be considered under control again?

In cases of outbreaks, food business operators face inspections, recall actions and delisting by retailers. This could have happened to an Austrian meat processor whose products have been associated with a cluster of seven cases of listeriosis spread over the years 2015-2017. Sequencing of clinical and foodborne isolates by public health specialists raised the suspect of a single source outbreak since all strains were of MLST 155, cgMLST 1234. Since the family-driven business was highly motivated to save their business, a crisis management scheme was applied that was agreed upon with national authorities. An end-product-based approach testing every single lot for L. monocytogenes was set into power and only negative lots were released for delivery. We combined the active food lot controls of food authorities with a Listeria environmental transmission mapping procedure. The environmental monitoring approach included 19 sampling activities during 3.5 years resulting in 1632 samples. This scheme allowed to trace and mitigate the Listeria contamination but did not jeopardize the processing of meat products. In total, 14 measures were set into power that reduced the overall Listeria occurrence after sanitation of 50-75 % (sampling event I, II) to 0.0-3.8 % (sampling events XIII to XIX). The outbreak-associated ST155/CT1234 clone was not detected in the third sampling event onwards but popped up during the sampling event VIII again. From then on, the outbreak clone ST155/CT1234 was no longer detected in the food business operator (FBO). We conclude that an intense combined investigation of food lots and environmental samples is needed to identify the source and verify that contamination levels are under control. Initially public health authorities suspected contamination of the slicer, but the monitoring approach has localized the source of ST155/CT1234 in a Schnitzel sorting machine. Other factors leading to the contamination scenario were inadequate conveyor belt hygiene. An inadequate crate washing system and an inadequate hygiene lock led to Listeria spreading between compartments. All transmission routes could be effectively interrupted. A root cause analysis and preventive maintenance program implemented in the FPE is mandatory for food processing facilities.

Liquid chromatography-tandem mass spectrometry and confirmation by liquid chromatography-high-resolution mass spectrometry hair tests to evidence use of tizanidine by racing cyclists.

Tizanidine, an imidazoline derivative close to clonidine, is a central alpha-2 adrenergic receptor agonist. Therapeutically, the drug is used as a muscle relaxant under the trade names Sirdalud™ or Zanaflex™. The drug is not prohibited by the World Anti-Doping Agency but, for therapeutic purposes, can only be obtained via a nominative temporary use authorization. The French public health police requested the laboratory to test for tizanidine in head hair specimens collected from international racing cyclists. Using Liquid chromatography-tandem mass spectrometry (LC/MS-MS) and confirmation by liquid chromatography-high-resolution mass spectrometry (LC/HRMS), after pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction, tizanidine was identified in the hair of three athletes at 1.1, 3.7, and 11.1 pg/mg. This is the first evidence that tizanidine is incorporated in human hair. However, it was not possible to interpret the data in terms of doses and frequency of use due to a lack of controlled study.

[Changes in Test Methods for Internationalization in the Japanese Pharmacopoeia (Part 1): Establishment of a Quantitative Test Method for Clonidine Hydrochloride Using HPLC Analysis].

The Japanese Pharmacopoeia (JP) is an official normative guide for maintaining the authenticity of properties and qualities of medicine in Japan. The JP is revised every 5 years, and partial amendments are made from time to time to keep abreast with progress in science and technology and international harmonization. We are conducting a related study on the elimination of toxic reagents from the JP. The elimination of toxic reagents is an important study in relation to the five pillars of the revision of the 18th JP, "Improvement in quality by proactively introducing the latest knowledge and technological advances". In addition, "Internationalization of the JP" is an important issue to be addressed during revision of the JP. Considering international harmonization of the JP, it is important to incorporate the test methods that have been used in other pharmacopoeia, such as the United States Pharmacopeia (USP) and the European Pharmacopoeia (EP) in the JP. To achieve the above, herein, we selected clonidine hydrochloride, which is listed in the 17th JP. A potentiometric titration method is used as a quantitative method for clonidine hydrochloride in the 17th JP; in contrast, a HPLC method is utilized in the USP and the EP. In this study, we synthesized impurities of clonidine hydrochloride and determined their purities using quantitative NMR. In addition, the complete separation conditions of these compounds by HPLC were examined, and simultaneous analysis was performed.

Thermoreversible Switchlike Electrocatalytic Reduction of Tizanidine Based on a Graphene Oxide Tethered Stimuli-Responsive Smart Surface Supported Pd Catalyst.

In this work, a graphene oxide (GRO)-based temperature-sensitive smart catalytic support material was developed by tethering biodegradable and hydrophilic poly(N-vinylcaprolactam) (PVCL) on a GRO (i.e., GRO-PVCL) surface. GRO-PVCL-supported palladium catalyst (i.e., Pd/GRO-PVCL) was then prepared for tizanidine (TZN) electroreduction. The impact of a temperature-sensitive smart surface on the electrochemical and electrocatalytic properties was examined. Moreover, when the large surface area, excellent electron transfer, and electrochemical catalysis abilities of GRO were combined with the responsive characteristics of PVCL, temperature-triggered reversible electrocatalysis of TZN with enhanced sensitivity has been proved. Results designated that GRO-PVCL exposed the hydrophilic surface at 20 °C, resulting in Pd NPs highly dispersed on the GRO-PVCL surface. Subsequently, the wettability of the Pd catalyst surface arbitrarily adapted to hydrophobicity at 40 °C, which highly enhanced the TZN reduction on the catalyst in electrochemical detection. The synergistic effect amid Pd and GRO-PVCL on Pd/GRO-PVCL improved the electrocatalytic activity of TZN. The detection of TZN with the Pd/GRO-PVCL modified electrode ranged from 0.02 to 276 µM with a low detection limit of 0.0015 µM at 40 °C. The Pd/GRO-PVCL modified electrode also possesses excellent stability, reproducibility, and anti-interference ability. Lastly, the modified electrode attained good recovery results in human urine and human plasma samples for the determination of TZN and also pharmacokinetics study in rat plasma.

Toward Higher Sensitivity in Quantitative MALDI Imaging Mass Spectrometry of CNS Drugs Using a Nonpolar Matrix.

Tissue-specific ion suppression is an unavoidable matrix effect in MALDI mass spectrometry imaging (MALDI-MSI), the negative impact of which on precision and accuracy in quantitative MALDI-MSI can be reduced to some extent by applying isotope internal standards for normalization and matrix-matched calibration routines. The detection sensitivity still suffers, however, often resulting in significant loss of signal for the investigated analytes. An MSI application considerably affected by this phenomenon is the quantitative spatial analysis of central nervous system (CNS) drugs. Most of these drugs are low molecular weight, lipophilic compounds, which exhibit inefficient desorption and ionization during MALDI using conventional polar acidic matrices (CHCA, DHB). Here, we present the application of the (2-[(2 E)-3-(4- tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile) matrix for high sensitivity imaging of CNS drugs in mouse brain sections. Since DCTB is usually described as an electron-transfer matrix, we provide a rationale (i.e., computational calculations of gas-phase proton affinity and ionization energy) for an additional proton-transfer ionization mechanism with this matrix. Furthermore, we compare the extent of signal suppression for five different CNS drugs when employing DCTB versus CHCA matrices. The results showed that the signal suppression was not only several times lower with DCTB than with CHCA but also depended on the specific tissue investigated. Finally, we present the application of DCTB and ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry to quantitative MALDI imaging of the anesthetic drug xylazine in mouse brain sections based on a linear matrix-matched calibration curve. DCTB afforded up to 100-fold signal intensity improvement over CHCA when comparing representative single MSI pixels and >440-fold improvement for the averaged mass spectrum of the adjacent tissue sections.

A comparative study of sterically and electro-statically stabilized silver nanoparticles for the determination of muscle relaxant tizanidine: Insights of localized surface plasmon resonance, surface enhanced Raman spectroscopy and electrocatalytic activity.

A comparative study of two types of silver nanoparticles was investigated. The effect of the surface chemistry of the studied silver nanoparticles (AgNPs) on their localized surface plasmon resonance was utilized for the quantitative determination of muscle relaxant tizanidine drug. The studied AgNPs are classified according to the type of stabilizing agent used in their synthesis into electrostatically and sterically stabilized AgNPs. The electrostatically-stabilized AgNPs (AA AgNPs) were prepared using ascorbic acid as both reducing and protective agents in alkaline medium. The sterically-stabilized AgNPs type (PEG-AA AgNPs) was prepared using ascorbic acid as a reducing agent and polyethylene glycol as a stabilizing agent at room temperature. The interaction of tizanidine with AgNPs was characterized using four different techniques including, transmission electron microscope, UV-visible spectrophotometric, surface enhanced Raman spectroscopic (SERS) and electroanalytical methods. SERS method was developed to study the relationship between the plasmon resonance and the enhanced power of Raman signal. The electrocatalytic behavior and the interfacial properties of AgNPs were studied using cyclic voltammetry and electrochemical impedance spectroscopy (EIS) on glassy carbon electrode modified with AgNPs. The quantitative determination of tizanidine in pharmaceutical and biological samples was successfully achieved by using AgNPs probe based on spectrophotometric methods. A linear response over the range 10-400 nmol L-1 was obtained. Validation procedures were carried out following International Conference on Harmonization (ICH) guidelines.

Simultaneous Determination of Tizanidine, Nimesulide, Aceclofenac and Paracetamol in Tablets and Biological Fluids Using Micellar Liquid Chromatography.

A simple, sensitive and rapid micellar liquid chromatographic method was developed and validated for simultaneous determination of four drugs, namely, paracetamol (PAR), tizanidine (TZD), aceclofenac (ACF) and nimesulide (NMD). Good chromatographic separation was achieved using Cyano column and micellar mobile phase consisting of 120 mM sodium dodecyl sulfate, 25 mM phosphate buffer and 10% (V/V) butanol. The pH was adjusted to three using phosphoric acid. The total retention time was below 10 min. The analysis was performed at a flow rate of 1 mL/min and a column temperature of 40°C with direct UV detection at 230 nm. Diclofenac sodium was used as the internal standard. The proposed method was validated according to the ICH guidelines and was successfully applied to the analysis of these drugs in their tablet dosage forms with high accuracy. Limits of detection were found to be 0.03, 0.07, 0.033 and 0.11 µg/mL for PAR, ACF, TZD and NMD, respectively. The high sensitivity of developed method permitted its application to the in-vitro determination of the cited drugs in spiked human plasma and urine samples, and the obtained results were satisfactory. However, PAR could not be determined in spiked human urine because its peak overlapped with that of the urine peak.

Identification of Drugs in Parenteral Pharmaceutical Preparations from a Quality Assurance and a Diversion Program by Direct Analysis in Real-Time AccuTOFTM-Mass Spectrometry (DART-MS).

In healthcare settings drug diversion and impairment of physicians are major concerns requiring a rapid and efficient method for surveillance and detection. A Direct Analysis in Real Time ion source coupled to a JEOL AccuTOFTM time-of-flight mass spectrometer (DART-MS) method was developed to screen parenteral pharmaceutical formulations for potential drug diversion. Parenteral pharmaceutical formulations are also known as injectable formulations and are used with intravenous, subcutaneous, intramuscular and intra-articular administration. A library was created using the mass spectra data collected by a DART-MS operated in switching mode at 20, 60 and 90 V settings. This library contained 17 commonly encountered drugs in parenteral pharmaceutical formulations that included the surgical analgesic: fentanyl, hydromorphone and morphine; anesthetic: baclofen, bupivacaine, ketamine, midazolam, ropivacaine and succinylcholine; and a mixture of other drug classes: caffeine, clonidine, dexamethasone, ephedrine, heparin, methadone, oxytocin and phenylephrine. Randomly selected 200 de-identified parenteral pharmaceutical formulations containing one or more drugs were submitted for analysis to the FIRM Toxicology Laboratory at Virginia Commonwealth University Health and were screened using the DART-MS. The drug contents of the de-identified formulations were previously confirmed by a published high performance liquid chromatography (HPLC) method. The drugs in the formulations were rapidly and successfully identified using the generated library. The DART-MS and HPLC results were in complete agreement for all 200 parenteral pharmaceutical formulations.

Quantitative target and systematic non-target analysis of polar organic micro-pollutants along the river Rhine using high-resolution mass-spectrometry--Identification of unknown sources and compounds.

In this study, the contamination by polar organic pollutants was investigated along the Rhine River, an important source of drinking water for 22 million people in central Europe. Following the flow of the river, a traveling water mass was sampled using weekly flow-proportional composite samples at ten different downstream sites, including main tributaries. Using a broad analytical method based on solid phase extraction and high-resolution mass spectrometry, the water was analyzed for more than 300 target substances. While the water in Lake Constance contained only 83 substances in often low concentrations, the number of detects found in the water phase increased to 143 substances and a weekly load of more than 7 tons at the last sampling site, the Dutch-German border. Mostly present were chemicals originating from wastewater treatment plants, especially the artificial sweetener Acesulfam and two pharmaceuticals, Metformin and Gabapentin, which dominate the weekly load up to 58%. Although the sample campaign was performed in a dry period in early spring, a large variety of pesticides and biocides were detected. Several industrial point sources were identified along the waterway's 900 km journey, resulting in high concentrations in the tributaries and loads of up to 160 kg. Additionally, an unbiased non-target analysis was performed following two different strategies for the prioritization of hundreds of potentially relevant unknown masses. While for the first prioritization strategy, only chlorinated compounds were extracted from the mass spectrometer datasets, the second prioritization strategy was performed using a systematic reduction approach between the different sampling sites. Among others, two substances that never had been detected before in this river, namely, the muscle relaxant Tizanidine and the solvent 1,3-Dimethyl-2-imidazolidinone (DMI), were identified and confirmed, and their loads were roughly estimated along the river.

Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations, human plasma and urine through derivatization with dansyl chloride.

A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the ranges 50-500 and 20-300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t- and F-tests.

Ability to estimate relative percutaneous penetration via a surrogate maker - trans epidermal water loss?

AIMS: This study measures the dynamic change of the trans-epidermal water loss (TEWL) rate and in vitro skin permeation data of tritiated water and [(14) C]-clonidine HCl in order to refine our knowledge in the relationship between percutaneous penetration and TEWL. MEASURES: TEWL values were measured before and during the experimental period. Single application of tritiated water and [(14) C]-clonidine HCl were dosed at the same time on dermatomed human skin samples collected from 12 donors in a flow through diffusion cell system. Radioactivity of absorbed dose: stratum corneum, epidermis, dermis, receptor fluid collected every 4 hours, as well as removable dose residue was counted to determine accountability, percent dose, µg equivalent, and flux rate. These data were further combined with TEWL values to analyze their possible relationship. RESULTS: Results showed that baseline TEWL values correlated with the thickness of dermatomed skin (r=-0.44, P=0.007), and with tritiated water fluxes (r=0.34, P=0.04) and [(14) C]-clonidine HCl (r=0.36; P=0.03). The fluxes of tritiated water and [(14) C]-clonidine HCl were correlated (r=0.67, P<0.001). When TEWL and permeation data were compared, the pattern of tritiated water expressed as a percent dose permeated in receptor fluid resembled the TEWL pattern. CONCLUSION: The methodology described provides evidences of the correlation of TEWL and skin integrity and skin permeation and further demonstrates to be a rapid alternative to tritiated water permeation for measuring skin barrier functions in vitro. To develop TEWL measurement as a possible predictive model to assess in vitro percutaneous absorption, however more chemicals with various physical-chemical properties need to be examined, and the relationships to TEWL and tritiated water flux better defined.

Development and validation of stability-indicating HPLC method for the simultaneous determination of paracetamol, tizanidine, and diclofenac in pharmaceuticals and human serum.

A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections.

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 microm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P(oct) measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as beta-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-microm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application.

Multiplexing G protein-coupled receptors in microarrays: a radioligand-binding assay.

Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of "high throughput" or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the alpha-adrenergic receptor family, alpha2A and alpha2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio approximately 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed.

Sensitive and convenient method for the quantification of clonidine in serum of pediatric patients using liquid chromatography/tandem mass spectrometry.

An improved and easy to use liquid chromatography/tandem mass spectrometric (LC/MS/MS) method in human serum was developed for the quantification of clonidine (CLD), an alpha2-/alpha1-adrenoceptor agonist, used for analgo-sedation and the therapy of opioid withdrawal in pediatric patients. Sample preparation consisted of precipitation of serum proteins by adding acetonitrile and centrifugation of the sample subsequently. [(2)H4]Clonidine (CLD4) served as internal standard. Chromatographic separation of the supernatant was achieved using a 100mmx3mm, 5microm Thermo Electron BetaBasic C4 column with isocratic flow and elution consisting of 0.1% formic acid/acetonitrile (85/15, v/v) and a flow-rate of 350microl/min resulting in a column pressure of 280-420kPa. LC/MS/MS detection was performed by using a triple-stage quadrupole mass spectrometer (TSQ Quantum, Thermo Electron) working in selected reaction monitoring mode with positive electrospray ionization. The analyte was quantified in a single run within 5min. Linearity was demonstrated over the expected concentration range 0.15-50microg/l CLD. The lower limit of quantification (LLOQ) and the limit of detection were 0.1microg/l and 0.01microg/l, respectively. None of the drugs used concomitantly during analgesic therapy interfered in the assay in vitro. Intra-day precision expressed as RSD was 9.6% or less for CLD, while inter-day result was 10.0% or less for CLD. Intra-day and inter-day accuracy was within +/-4.9% and +/-1.8%, respectively. The method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the US Food and Drugs Administration (FDA). The described method is suitable to analyse serum samples with very small volumes and sets the stage for pharmacokinetic studies in pediatric studies.

In vitro and in vivo characterization of a newly developed clonidine transdermal patch for treatment of attention deficit hyperactivity disorder in children.

The aim of this study was to characterize a newly developed clonidine transdermal patch, KBD-transdermal therapeutic system (TTS), for the treatment of attention deficit hyperactivity disorder in children. In vitro release, penetration, and in vivo pharmacokinetics in rabbits were investigated. The smaller size of KBD-TTS (2.5 mg/2.5 cm2) showed a similar in vitro penetration to those of Catapres-TTS (2.5 mg/3.5 cm2, a clonidine transdermal patch used for the treatment of hypertension, Alza Corporation, U.S.A.). The transdermal penetration rate of clonidine was mainly controlled by the ethylene vinylacetate membrane used in the patch. The skin layer may be only a minor rate-limiting barrier after the topical skin layer at the dosing site is saturated with penetrating clonidine in the initial phase (0 to 12 h). A sensitive liquid chromatography-mass spectrometry method for the quantification of clonidine in rabbit plasma was developed using solid-phase extraction and gradient elution on LC combined with the selected-ion monitoring (SIM) mode. A single dose of clonidine transdermal patch (KBD-TTS) or Catapres-TTS was transdermally administered to rabbits (n=6 each) and removed after 168 h. The average half-life, Tmax, Cmax and Css values of clonidine in rabbits following administration of KBD-TTS were 19.27+/-4.68 h, 52.56+/-25.77 h, 27.39+/-9.03 ng/ml, and 25.82+/-9.34 ng/ml, similar to those of Catapres-TTS, respectively. The clonidine plasma concentration of KBD-TTS reached a steady state at 24 h through 168 h. The in vitro release rate of the clonidine from KBD-TTS significantly correlated with the in vivo absorption rate (p<0.001).

Application of HPLC and HPTLC for the simultaneous determination of tizanidine and rofecoxib in pharmaceutical dosage form.

Two methods are described for the simultaneous determination of tizanidine and rofecoxib in binary mixture. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 311 nm. The separation was carried out on Merck HPTLC aluminium sheets of silica gel 60 F254 using toluene:methanol:acetone (7.5:2.5:1.0, v/v/v) as mobile phase. The linear regression analysis data was used for the regression line in the range of 10-100 and 100-1500 ng/spot for tizanidine and rofecoxib, respectively. The second method was based on HPLC separation of the two drugs on the reversed phase kromasil column [C18 (5 microm, 25 cm x 4.6 mm, i.d.)] at ambient temperature using a mobile phase consisting of phosphate buffer pH 5.5 and methanol (45:55, v/v). Flow rate was 1.0 ml/min with an average operating pressure of 180 kg/cm2. Quantitation was achieved with UV detection at 235 nm based on peak area with linear calibration curves at concentration ranges 10-200 and 100-2000 microg/ml for tizanidine and rofecoxib, respectively. Both methods have been successively applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found. Both methods were validated in terms of precision, robustness, recovery and limits of detection and quantitation. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of tizanidine and rofecoxib determination in dosage form by means of HPTLC and HPLC method.

Simultaneous LC determination of tizanidine and rofecoxib in tablets.

A reverse phase high performance liquid chromatographic method to determine tizanidine (TZ) and rofecoxib (RF) in combination is proposed and applied to the pharmaceuticals. This method allows the determination of 0.1-0.5 microg/ml of TZ and 1.2-6.0 microg/ml of RF along with 10 microg/ml of nimesulide (internal standard), in a mobile phase consisting of 1% (v/v) triethylamine (pH adjusted to 2.5 using dilute orthophosphoric acid):acetonitrile in the ratio 55:45% (v/v). Detection wavelength of 303 nm and flow rate of 0.8 ml/min were fixed for the study. The limit of detection (LOD) for TZ and RF were found to be 10 and 1 ng/ml, respectively. The limit of quantification (LOQ) for TZ and RF were found to be 80 and 12 ng/ml, respectively. The amount of drug present in the tablet and the recovery studies were also carried out. The % R.S.D. of recovery studies for TZ and RF were found to be 0.0673 and 0.0146, respectively. The method is validated for accuracy, precision, ruggedness and robustness.

Stability of clonidine in clonidine-hydromorphone mixture from implanted intrathecal infusion pumps in chronic pain patients.

Clonidine is frequently added to opioids in implantable intrathecal pumps for the management of chronic pain. In such devices, a small non-retrievable volume is always present in the reservoir, and its effect on drug stability is unknown. Furthermore, stability of clonidine, when mixed with hydromorphone, has not been previously determined. This study examined the stability of clonidine when co-administered with hydromorphone in implanted intrathecal pumps. Samples of hydromorphone-clonidine before pump refill and from residual solution at subsequent refill were obtained from chronic pain patients. Clonidine concentration was measured using HPLC. Twenty paired samples from 3 patients were analyzed. All 3 patients had a SynchroMed pump implanted for 3-5 years. We found no loss in clonidine concentration during the time between refills (35 +/- 13 days), and no correlation between clonidine concentration and time interval between refills. In conclusion, clonidine, mixed with hydromorphone, is stable when delivered by implantable intrathecal pump for long-term use.